In the field of biological analyses the importance of the determination and precise counting of different cell populations in making a diagnosis has been recognised for a long time. In fact, the appearance of abnormal equilibrium ratios among normal cell populations in blood may be correlated with the appearance of certain medical conditions, for example immune reactions, inflammatory reactions, etc. Similarly, the appearance of abnormal cell populations may also be correlated with the appearance of other conditions, such as leukaemia, etc.
There are various conventional methods of cytological analysis, involving microscopic examination after staining, and if necessary after sedimentation or aggregation. The automatic determination of blood cells began at the beginning of the 1960s with the separation of the main normal leucocyte populations; see the following bibliographic reference: (1) Hallerman L., Thom R., Gerhartz H.: “Elecktronische Differentialzählzung von Granulocyten und Lymphocyten nach intervaler Fluochromierung mit Acridinorange” (“Electronic Differential Counting of Granulocytes and Lymphocytes by Interval Fluorochrome Staining with Acridine Orange”), Verh Deutsch Ges Inn Med 70: 217, 1964.
The separation of the leucocytes was performed by flow cytometry utilising various principles involving the optical and chemical properties of the cells. Several automated haematology analysers have been produced, using various techniques such as Coulter's principle for determining volumes, measurement of diffracted light for estimating sizes, measurement of diffused light at 90° for determining the internal structures of cells, and fluorescence or absorption measurements for determining the affinities of cells for various stains; see the following bibliographic references 2 to 5:
(2) Adams L. R., Kamensky L. A.: “Fluorometric Characterization of Six Classes of Human Leukocytes” Acta Cytol 18: 389, 1974;
(3) Shapiro H. M. et al. “Combined Blood Cell Counting and Classification with Fluorochrome Stains and Flow Instrumentation” J. Histochem Cytochem 24: 396-41.1, 1976;
(4) Terstappen L. W. et al. “Multidimensional Flow Cytometric Blood Cell Differentiation Without Erythrocyte Lysis” Blood Cells 17: 585-602, 1991;
(5) Terstappen L. W., Levin J. “Bone marrow cell differential counts obtained by multidimensional flow cytometry” Blood Cells 18 (2): 311-30, 1992.
The characterisation of cells in early stages of the cell cycle has long been of interest to scientists and the quantification of the RNA content of each cell has for a long time been recognised as a representative parameter of this cycle; see the bibliographic references 2 to 5 above and the following bibliographic references 6 and 7:
(6) Traganos F., Darzynkiewicz Z., Sharpless T., Melamed M. R. “Simulataneous Staining of Ribonucleic and Deoxyribonucleic Acids in Unfixed Cells Using Acridine Orange in a Flow Cytofluorometric System”
J. Histochem Cytochem 25: 46, 1977; (7) Pollack A. et al. “Flow Cytometric Analysis of RNA Content in Different Cell Populations Using Pyronin Y and Methyl Green” Cytometry, vol. 3, no. 1, pages 28-35, 1982.
In their French patent no. 97 01090, dated 31 Jan. 1997, the Applicants have already described a composition, and more particularly a staining reagent, enabling this type of analysis to be performed.
In order to automate such techniques various problems first of all have to be solved, in particular reducing the treatment times and cost of preparing the samples. Such a reduction may be achieved in various ways, the most obvious being to reduce the number of channels so that only one cell preparation is carried out at any one time. This type of technique has previously been described by Léon W. Terstappen (reference 4 above), but requires a long treatment and analysis time, in particular for the accurate counting of the nucleate cells, the number of which is often a thousand times less than the number of erythrocytes.
In order to obviate this difficulty the biological sample is often separated into at least two aliquot parts, one of which is prepared at a certain concentration enabling the erythrocytes and platelets to be studied, the other being prepared at a higher concentration for the analysis of the nucleate cells.
These known techniques have various disadvantages.
Before the analysis, the treatment of this aliquot part often involves the specific destruction of the erythrocytes in order to facilitate the measurement of the remaining cells. Although such a method enables the results of the measurements to be obtained more quickly, this is nevertheless offset by the time involved in the reaction, transfer and staining in order to obtain the desired preparation.
The incubation time of a cell suspension in a reagent solution is in particular associated with the time required for the active principles to penetrate the interior of the cells. In French patent no. 97 01090 mentioned hereinbefore, the Applicants have described ways of accelerating this penetration involving the use of an additive, in particular an ionophore type additive, to assist the cell penetration.
The treatment time is also a function of the number of successive stages that the aliquot part has to pass through. Lysis and staining of the cells are often carried out in two successive stages, in one order or the other (see U.S. Pat. No. 6,004,816).
These two dilution stages involve a not inconsiderable expenditure in material, associated with a long minimum treatment time.